Association between neutrophil-to-lymphocyte ratio and diabetic retinopathy in patients with type 2 diabetes: a cohort study

Background Chronic inflammation is implicated in the development of diabetic retinopathy (DR). The neutrophil-to-lymphocyte ratio (NLR) is a marker of systemic inflammation that has been linked to cardiovascular and diabetic kidney diseases. However, the link between NLR and DR remains unclear. As such, this study investigated the association between NLR and DR in Chinese patients. Method A total of 857 adults diagnosed with type 2 diabetes mellitus (T2DM) without DR at baseline between 2018 and 2021, from a single center in Ningbo, China, were included. Baseline clinical data, including age, sex, T2DM duration, hypertension, smoking, drinking, glycated hemoglobin level, lipid profile, renal function, and NLR, were recorded and analyzed. Cox proportional hazard regression analysis was used to assess the association between NLR and the risk for incident DR. Results During a median follow-up of 3.0 years, 140 patients developed DR. The multivariable-adjusted hazard ratio (HR) for incident DR across ascending NLR quartiles (≤1.46 [reference], 1.47–1.90, 1.91–2.45 and > 2.45) were 1.000, 1.327 (95% confidence interval [CI] 0.754–2.334), 1.555 (95% CI 0.913–2.648) and 2.217 (95% CI 1.348–3.649), respectively. For each 1-standard deviation increase in NLR, the risk for DR increased by 29.2% (HR 1.292 [95% CI 1.112–1.501) after adjusting for confounding factors. Conclusion Results revealed that a higher NLR at baseline was associated with an increased risk for incident DR. NLR has the potential to be an inexpensive, reliable, and valuable clinical measure that merits further exploration in future studies.


Introduction
Diabetes mellitus (DM) is a slowly progressive metabolic illness characterized by long-standing hyperglycemia that can cause various complications (1).Diabetic retinopathy (DR) is one of the most prevalent complications and a leading cause of blindness among working-age adults, seriously affecting patient quality of life and increased financial burden (2, 3).The early detection and prevention of DR are crucial to avoid blindness.
There is accumulating evidence that chronic low-grade inflammation has a particular influence on the development of DM and metabolic syndrome (4).Chronic inflammation is a hallmark of type 2 DM (T2DM), possibly due to insulin resistance and impaired glucose metabolism (5).
DR is a progressive microvascular and neurodegenerative disease that occurs gradually with years of poor glycemic control (6).Although clinical signs, such as acellular capillaries, retinal nonperfusion, and ischemia appear later, early changes involve persistent low-grade leukocyte activation triggered by metabolic dysregulation (7).This activation disrupts the retinal neurovascular unit and its cellular components (7).
The neutrophil-to-lymphocyte ratio (NLR), which is the synthesis of two distinct but complementary immune pathways of the innate and adaptive cellular immune responses, has been studied extensively as an inflammatory marker in many malignant and benign diseases (8).Western studies have reported that an elevated NLR is independently linked to increased cardiovascular death and all-cause mortality in patients with diabetes, as well as a higher risk for diabetic kidney disease (9,10).However, the relationship between the NLR and DR remains unclear, with only a limited number of cross-sectional studies exploring this relationship (11,12).As such, the present cohort study aimed to evaluate whether the presence of DR is correlated with NLR in Chinese patients.

Study population
In this retrospective cohort study, patients diagnosed with T2DM, who attended the Department of Endocrinology and Metabolism, First Affiliated Hospital of Ningbo University, and participated in the multihospital-based program of the National Metabolic Management Center (MMC) were recruited from March 2018 to December 2021.The MMC program, led by Ruijin Hospital in Shanghai (China), followed a standard protocol for the professional management and follow-up of patients with DM (13).
The inclusion criteria were as follows: age 18-75 years; confirmed T2DM diagnosis (1999 World Health Organization criteria ( 14)); absence of DR at baseline; and available annual fundus photography data.Exclusion criteria were as follows: history of other fundus diseases; history of hematological diseases; acute diabetic complications, such as diabetic ketoacidosis; other concomitant conditions influencing glucose metabolism (acute infection, chemotherapy, use of corticosteroids); history of cancer, heart failure, end-stage renal disease, chronic liver disease; or loss to follow-up or short follow-up (<2 years).In total, data from 857 patients were included in the final analysis.This study adhered to the Declaration of Helsinki and was approved by the Ethics Committees of both hospitals (Ruijin Hospital: 2017 No. 42; Ningbo University: 2019-R057).Informed consent was obtained from all participants.

Baseline clinical and biological characteristics
Demographic information (age, sex, and T2DM duration), lifestyle habits (history of smoking and alcohol consumption), and medical histories were obtained using standardized MMC questionnaires.Trained nurses measured the height, weight, and waist circumference of the participants.Body mass index was calculated as the ratio of weight (kg) to height squared (m 2 ).Visceral fat area was assessed using dual bioelectrical impedance analysis (DUALSCAN HDS-2000, Omron, Japan).
After an overnight fast, venous blood and urine samples were collected in the morning.Complete blood count with differential analysis was performed using an automated analyzer (Sysmex XN-9000, Kobe, Japan).The NLR was calculated.Glycated hemoglobin (HbA1c), fasting plasma glucose, and fasting insulin levels were measured using established methods (D-10 Hemoglobin Analyzer, Bio-Rad, Hercules, CA, USA; glucose oxidase method; chemiluminescence immunoassay).The Homeostatic Model Assessment for Insulin Resistance (i.e., "HOMA-IR") index, reflecting insulin resistance, was calculated using a standard equation: fasting blood glucose (FBG, mmol/L) × fasting insulin (mU/L) ÷ 22.5 (17).Serum creatinine and uric acid levels were measured using standard enzymatic methods (AU5800, Beckman Coulter, Brea, CA, USA).The estimated glomerular filtration rate (eGFR) was determined using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) algorithm (18).The urinary albumin-to-creatinine ratio (UACR) was calculated from the ratio of urinary albumin to creatinine measured in spot urine samples using immunonephelometry (albumin) and enzymatic methods (creatinine).

Outcome
The primary outcome was incident DR.All patients underwent non-mydriatic fundus photography at the first visit and were reevaluated for DR during each annual visit (Topcon, Tokyo, Japan) (19).Two images were captured per eye, centered on the macula and optic nerve at 45°, following established protocols for effective DR screening (20).DR was diagnosed based on the International Classification of DR (21).

Statistical analysis
Normally distributed continuous variables are expressed as mean ± standard deviation (SD) and were compared using independent-samples t-tests.Continuous variables with nonnormal distribution are expressed as median and interquartile range (i.e., 25th percentile, 75th percentile) and compared using the Mann-Whitney U test.Categorical variables are expressed as frequency (percentage) and compared using the chi-squared test to assess differences in baseline characteristics between patients with incident DR and those without DR (NDR) in the T2DM group.

Discussion
The present cohort study identified a positive correlation between NLR, a composite metric for chronic inflammation, and the incidence of DR in patients with T2DM.Each 1-SD increase in the NLR corresponded to a 29% increased risk for developing DR.Chronic low-grade inflammation and endothelial dysfunction are well-established contributors to insulin resistance, T2DM, and other microvascular and macrovascular complications (12).Inflammatory responses triggered by hyperglycemia play a key role in vascular dysfunction.Hyperglycemia induces the production of reactive oxygen species and upregulates the expression of pro-inflammatory and pro-coagulant factors, which promote adhesion between white blood cells and endothelial cells (22).The accumulation of leukocytes in capillaries disrupts the normal structure between the retinal endothelial cells and pericytes, leading to a damaged blood-retinal barrier.Excessive accumulation of leukocytes can cause vascular blockage and leakage, which in turn exacerbate damage to the blood-retinal barrier (23).Overall, the emergence of inflammatory mediators and disturbances in the balance between angiogenesis-related factors promote the development of DR (24).This results in increased retinal vascular permeability, neurodegeneration, damage to the blood-reticulum barrier, and neovascularization, ultimately leading to diabetic macular edema and proliferative DR (24).NLR reflects both cellular immune activation and systemic inflammatory response, acting as an indicator of the balance between two key immune system components: neutrophils, active non-specific inflammatory cells, lymphocytes, and protective or regulatory cells (8).The NLR has emerged as a promising marker in this context.
Our results are consistent with those of most previous studies that have explored the relationship between the inflammatory index and diabetic complications.This aligns with a case-control study from Turkey (25), where NLR levels were higher in patients with diabetes and DR than in those without DR.While some studies, such as those from northern China (26), reported higher NLR in patients with DR, and the investigators did not find NLR to be an independent risk factor.Moreover, another cross-sectional study from Turkey that analyzed 114 patients with T2DM reported that NLR was not independently associated with DR (27).Potential explanations for these variations include the larger sample size, differences in subject characteristics, and variations in lifestyle factors.
Several studies have supported the link between NLR and diabetic microvascular complications.A cross-sectional study observed a positive association between a higher NLR and the occurrence of both nephropathy and retinopathy in an Indian population (12).Additionally, an elevated NLR was correlated with diabetic nephropathy in other studies (10,28) and even served as an independent predictor of kidney function decline in individuals with DM (29).DR and nephropathy share common underlying mechanisms and often co-occur (30).Given this shared etiology and the association between NLR and both complications, NLR may emerge as a valuable predictor and prognostic marker for both DR and nephropathy.
The present study had several limitations, the first of which was its retrospective cohort design, which precluded the determination of a causal relationship between the biomarkers and outcomes.Second, we did not analyze correlations stratified according to DR severity (i.e., non-proliferative and proliferative DR), because only 1 participant developed proliferative DR.Third, lifestyle, socioeconomic, and pharmacological interventions were not considered in this study.Additionally, the relatively short followup period limited our ability to fully assess the long-term impact of NLR on DR progression.Future studies with larger cohorts and extended follow-up periods are essential to validate our findings and explore these associations more thoroughly.
In summary, results of the present study indicate that the incidence of DR is associated with a higher NLR.These findings may provide instructive options for clinical practice.According to  statistics, there are an estimated 141 million patients with diabetes in China, and the number of patients with DR has exceeded 19 million (31,32).With an aging population and a high incidence of DM, dealing with visual impairment and blindness is a huge challenge.Screening for DR offers a preferable solution to decrease preventable blindness and the medical economic burden.
According to these guidelines, routine screening every to 1-2 years is recommended.Based on the results of our study, we recommend that patients with diabetes and higher NLR should undergo a shorter period of regular screening for DR (e.g., 6 months).Eye examination by fundus photography using a non-mydriatic fundus camera costs < $2 in our hospital, and early detection of DR could save thousands from medical expenditures for the treatment of DR.

Conclusion
In conclusion, results of the present study indicated that, among patients with T2DM, a higher NLR was associated with an increased risk for incident DR.Given the advantages of NLR in terms of stability and accessibility, this result further supports that NLR could be a valuable clinical measure.Future studies with various populations and designs are necessary to further explore the potential value of the NLR in predicting diabetes-related outcomes.

TABLE 1
Clinical characteristics of patients grouped by the incident diabetic retinopathy.

TABLE 2
Hazard ratios for diabetic retinopathy according to neutrophil-to-lymphocyte ratio as categorical variable.Model 2: adjusted for age and sex; Model 3: adjusted for age, sex, diabetes course, smoking history, drinking history, body mass index, systolic blood pressure, triglycerides, glycated hemoglobin, fasting blood glucose, fasting insulin, uric acid, glomerular filtration rate and urinary albumin-to-creatinine ratio.DR, diabetic retinopathy; NLR, neutrophil-to-lymphocyte ratio; HR, hazard ratio; CI, confidence interval.